Avital-Shmilovici Michal, Whittaker Jonathan, Weiss Michael A, Kent Stephen B H
From the Departments of Chemistry, and Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, University of Chicago, Chicago, Illinois 60637 and.
the Departments of Biochemistry and.
J Biol Chem. 2014 Aug 22;289(34):23683-92. doi: 10.1074/jbc.M114.572040. Epub 2014 Jul 7.
Misfolding of proinsulin variants in the pancreatic β-cell, a monogenic cause of permanent neonatal-onset diabetes mellitus, provides a model for a disease of protein toxicity. A hot spot for such clinical mutations is found at position B8, conserved as glycine within the vertebrate insulin superfamily. We set out to investigate the molecular basis of the aberrant properties of a proinsulin clinical mutant in which residue Gly(B8) is replaced by Ser(B8). Modular total chemical synthesis was used to prepare the wild-type [Gly(B8)]proinsulin molecule and three analogs: [D-Ala(B8)]proinsulin, [L-Ala(B8)]proinsulin, and the clinical mutant [L-Ser(B8)]proinsulin. The protein diastereomer [D-Ala(B8)]proinsulin produced higher folding yields at all pH values compared with the wild-type proinsulin and the other two analogs, but showed only very weak binding to the insulin receptor. The clinical mutant [L-Ser(B8)]proinsulin impaired folding at pH 7.5 even in the presence of protein-disulfide isomerase. Surprisingly, although [L-Ser(B8)]proinsulin did not fold well under the physiological conditions investigated, once folded the [L-Ser(B8)]proinsulin protein molecule bound to the insulin receptor more effectively than wild-type proinsulin. Such paradoxical gain of function (not pertinent in vivo due to impaired secretion of the mutant insulin) presumably reflects induced fit in the native mechanism of hormone-receptor engagement. This work provides insight into the molecular mechanism of a clinical mutation in the insulin gene associated with diabetes mellitus. These results dramatically illustrate the power of total protein synthesis, as enabled by modern chemical ligation methods, for the investigation of protein folding and misfolding.
胰岛素原变体在胰腺β细胞中错误折叠是永久性新生儿糖尿病的单基因病因，为蛋白质毒性疾病提供了一个模型。在B8位点发现了此类临床突变的一个热点，该位点在脊椎动物胰岛素超家族中保守为甘氨酸。我们着手研究一种胰岛素原临床突变体异常特性的分子基础，该突变体中残基甘氨酸（B8）被丝氨酸（B8）取代。采用模块化全化学合成法制备野生型[甘氨酸（B8）]胰岛素原分子和三种类似物：[D-丙氨酸（B8）]胰岛素原、[L-丙氨酸（B8）]胰岛素原和临床突变体[L-丝氨酸（B8）]胰岛素原。与野生型胰岛素原和其他两种类似物相比，蛋白质非对映异构体[D-丙氨酸（B8）]胰岛素原在所有pH值下产生的折叠产率更高，但与胰岛素受体的结合非常弱。临床突变体[L-丝氨酸（B8）]胰岛素原即使在存在蛋白质二硫键异构酶的情况下，在pH 7.5时也会损害折叠。令人惊讶的是，尽管[L-丝氨酸（B8）]胰岛素原在所研究的生理条件下折叠不佳，但一旦折叠，[L-丝氨酸（B8）]胰岛素原蛋白分子与胰岛素受体的结合比野生型胰岛素原更有效。这种矛盾的功能获得（由于突变胰岛素分泌受损，在体内不相关）可能反映了激素-受体结合天然机制中的诱导契合。这项工作为与糖尿病相关的胰岛素基因突变的分子机制提供了见解。这些结果显著说明了现代化学连接方法实现的全蛋白合成在研究蛋白质折叠和错误折叠方面的强大作用。
